Dialkyl ureas as calcitonin mimetics

ABSTRACT

Dialkyl urea compounds are described which act as calcitonin mimetics. These compounds are useful in the treatment of diseases which are associated with bone resorption. The calcitonin mimetics of the present invention are also useful in assays for the determination of calcitonin receptor activity.

This is a divisional application of application Ser. No. 09/233,893,filed on Jan. 20, 1999 which claims priority from ProvisionalApplication 60/072,987 filed on Jan. 21, 1998.

BACKGROUND OF THE INVENTION

Bone is a dynamic tissue, and homeostasis in the adult skeleton requiresa balance between bone resorption and bone formation. Osteoclasts andosteoblasts play a key role in this balance, with osteoclasts initiatingbone resorption and osteoblasts synthesizing and depositing new bonematrix. Imbalances in bone homeostasis are associated with suchconditions as osteoporosis, Paget's disease, and hyperparathyroidism.

The activities of osteoclasts and osteoblasts are regulated by complexinteractions between systemic hormones and the local production ofgrowth factors and cytokines. Calcitonin, a peptide hormone secreted bythe thyroid and thymus of mammals, plays an important role inmaintaining bone homeostasis. Calcitonin inhibits bone resorptionthrough binding and activation of a specific calcitonin receptor onosteoclasts (The Calcitonins-Physiology and Pharmacology Azria (ed.),Karger, Basel, Su., 1989), with a resultant decrease in the amount ofcalcium released by bone into the serum. This inhibition of boneresorption has been exploited, for instance, by using calcitonin as atreatment for osteoporosis, a disease characterized by a decrease in theskeletal mass often resulting in debilitating and painful fractures.Calcitonin is also used in the treatment of Paget's disease where itprovides rapid relief from bone pain, which is frequently the primarysymptom associated with this disease. This analgesic effect has alsobeen demonstrated in patients with osteoporosis or metastatic bonedisease and has been reported to relieve pain associated with diabeticneuropathy, cancer, migraine and post-hysterectomy. Reduction in bonepain occurs before the reduction of bone resorption.

Salmon calcitonin has been shown to be considerably more effective inarresting bone resorption than human forms of calcitonin. Severalhypotheses have been offered to explain this observation: 1) salmoncalcitonin is more resistant to degradation; 2) salmon calcitonin has alower metabolic clearance rate (MCR); and 3) salmon calcitonin may havea slightly different conformation, resulting in a higher affinity forbone receptor sites.

Despite the advantages associated with the use of salmon calcitonin inhumans, there are also disadvantages. For treatment of osteoporosis, forinstance, the average cost can exceed $75 a week and involve dailyprophylactic administration for 5 or more years. In the United States,calcitonin must be administered by injection, and since the diseaseindications for this drug are not usually life threatening, patientcompliance can be low. Resistance to calcitonin therapy may occur withlong-term use. What triggers this resistance or “escape phenomenon” isunknown (see page 1093, Principles of Bone Biology, Bilezikian et al.,(eds.) Academic Press, NY; Raisz et al., Am. J. Med. 43:684-90, 1967;McLeod and Raisz, Endocrine Res. Comm.8:49-59, 1981; Wener et al.,Endocrinology. 90:752-9, 1972 and Tashjian et al., Recent Prog. Horm.Res. 34:285-303, 1978). Use of calcitonin mimetics, either in place ofnative calcitonins or in rotation with native calcitonins, would helpavoid resistance to such treatment during long-term use. In addition,some patients develop antibodies to non-human calcitonin, calcitoninmimetics would be useful for such patients.

What is needed in the art are alternative methods of inhibiting boneresorption. The present invention fulfills these and other needs.

SUMMARY OF THE INVENTION

The present invention provides isolated compounds that are useful ascalcitonin mimetics. As used herein, the term “calcitonin mimetic”refers to a compound with the ability to mimic the effects generated bycalcitonin's interaction with its receptor and its signal transductionpathway and, by such interaction, stimulate G-protein-mediatedactivation by adenyl cyclase.

Within one aspect the invention provides a compound of formula I:

wherein R1 and R2 are each members independently selected from the groupconsisting of hydrogen, alkyls having from 1 to 6 carbon atoms, alkenylshaving from 1 to 6 carbon atoms, aryl, substituted aryl, alkylaryl,substituted alkylaryl, carbocyclic ring, substituted carbocyclic ring,heterocyclic ring, substituted heterocyclic ring, and combinationsthereof, the combinations are fused or covalently linked and thesubstituents are selected from the group consisting of halogen,haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino,monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl; R3 is a 2,5disubstituted aryl; R4 and R5 are each independently selected from thegroup consisting of hydrogen and alkyls having from 1 to 6 carbon atoms,or taken together from a ring selected from the group consisting ofsaturated or unsaturated five-member rings, saturated or unsaturatedsix-member rings and saturated or unsaturated seven-member rings; Z andX are each independently selected from the group NH, O, S, or NR,wherein R is a lower alkyl group of from 1 to 6 carbon atoms; n and mare each independently an integer from 0 to 6. Within one embodiment R1is selected from the group consisting of phenyl, substituted phenyl,benzyl, substituted benzyl, naphthylmethyl, substituted naphthylmethyl,indolymethyl, and substituted indolymethyl; R2 is selected from thegroup consisting of alkyls of from 1 to 6 carbon atoms, alkenyls of from1 to 6 carbon atoms, benzyl, substituted benzyl, naphthylmethyl, andsubstituted naphthylmethyl; wherein substituents are selected from thegroup consisting of halogen, haloalkyl, hydroxy, aryloxy, benzyloxy,alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl,alkyl and aryl; and R4 and R5 are hydrogen; Z is 0; and X is NH. Withina related embodiment R1 is 4-ethoxybenzyl, 1-ethyl-indolylmethyl,benzyl, 4-alloxybenzyl, 1-allyl-indolylmethyl, 4-chlorobenzyl,.4-flurobenzyl, 4-iodobenzyl, 2-naphthylmethyl or phenyl; and R2 isethyl, allyl, benzyl or 2-naphthylmethyl. Within another embodiment thecompound has the formula:

wherein, R1 and R2 are each independently selected from the groupconsisting of hydrogen, alkyls having from 1 to 6 carbon atoms, alkenylshaving from 1 to 6 carbon atoms, aryl, substituted aryl, alkylaryl,substituted alkylaryl, carbocyclic ring, substituted carbocyclic ring,heterocyclic ring, substituted heterocyclic ring, and combinationsthereof, the combinations are fused or covalently linked and thesubstituents are selected from the group consisting of halogen,haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino,monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl; and S1, S3and S4 are each independently selected from the group consisting ofhydrogen, halogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy,haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl, alkyland aryl. S2 and S5 are each independently alkyl or aryl. Within oneembodiment R1 is selected from the group consisting of phenyl,substituted phenyl, benzyl, substituted benzyl, naphthylmethyl,substituted naphthylmethyl, indolymethyl, and substituted indolymethyl;R2 is selected from the group consisting of alkyls having from 1 to 6carbon atoms, alkenyls having from 1 to 6 carbon atoms, benzyl,substituted benzyl, naphthylmethyl, and substituted naphthylmethyl;wherein the substituents are selected from the group consisting ofhalogen, haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy,amino, monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl andS2 and S5 are t-butyl. Within a related embodiment R1 is 4-ethoxybenzyl,1-ethyl-indolylmethyl, benzyl, 4-alloxybenzyl, 1-allyl-indolylmethyl,4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl, 2-naphthylmethyl or phenyl;R2 is ethyl, allyl, benzyl or 2-naphthylmethyl; and S2 and S5 aret-butyl.

Within another aspect, the invention provides a pharmaceuticalcomposition comprising an effective amount of a compound as describedabove in a pharmaceutically acceptable carrier.

Within another aspect the invention provides a method for treating abone-related disorder, comprising administering to a subject sufferingfrom such disorder an effective amount of calcitonin mimetic compound asdescribed above. Within a related embodiment the bone-related disorderis selected from the group consisting of osteoporosis, Paget's disease,hyperparathyroidism, osteomalacia, periodontal applications (bone loss),hypercalcemia of malignancy and hypercalcemia of infancy.

Within another aspect the invention provides a method of inhibiting boneresorption comprising administering to a subject in need of suchinhibition an effective amount of a calcitonin mimetic compound asdescribed above.

Within yet another aspect the invention provides a method for providingan analgesic effect comprising administering to a subject in need ofsuch an effect an effective amount of a calcitonin mimetic compound asdescribed above. Within a related embodiment the analgesic effectprovides relief from bone pain.

Within another aspect the invention provides a method for treatingconditions associated with inhibiting gastric secretion comprisingadministering to a subject in need of such inhibition an effectiveamount of a calcitonin mimetic compound as described above. Within arelated embodiment the conditions associated with inhibiting gastricsecretion is a gastrointestinal disorder.

These and other aspects of the invention will become evident uponreference to the following detailed description and the attacheddrawings.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations

The following abbreviations are used herein: Boc, t-butoxycarbonyl; DCM,dichloromethane; DME, dimethoxyethane; DMF, dimethylformamide; EtOAc,ethyl acetate; Fmoc, fluorenylmethoxycarbonyl; TFA, trifluoroaceticacid.

All references cited herein are incorporated by reference in theirentirety.

The calcitonin mimetics which are useful in the present invention arethose compounds with the ability to mimic the interaction of calcitoninwith its receptor and, by such mimicry, to stimulate G-protein-mediatedactivation of adenyl cyclase or activation of CRE by an alternativesignal transduction pathway. These mimetics are represented by thegeneral formula:

In this formula, R1 and R2 are each independently hydrogen, alkyl groupshaving from 1 to 6 carbon atoms, alkenyl groups having from 1 to 6carbon atoms, an aryl group, or alkylaryl groups, where the alkylportion may have 1 to 6 carbon atoms and the aryl portion represents anaryl group, a substituted aryl group, a carbocyclic ring, a substitutedcarbocyclic ring, a heterocyclic ring, a substituted heterocyclic ring,or combinations thereof. The combinations can be fused or covalentlylinked. In certain preferred embodiments R1 is substituted orunsubstituted phenyl, benzyl, naphthylmethyl or indolymethyl. R2 is analkyl or alkenyl having from 1 to 6 carbon atoms, substituted orunsubstituted benzyl or naphthylmethyl. In certain particularlypreferred embodiments R1 is 4-ethoxybenzyl, 1-ethyl-indolylmethyl,benzyl, 4-alloxybenzyl, 1-allyl-indolylmethyl, 4-chlorobenzyl,4-flurobenzyl, 4-iodobenzyl, 2-naphthylmethyl or phenyl. R2 is ethyl,allyl, benzyl, or 2-naphthylmethyl.

R3 represents substituted and unsubstituted aryl groups, carbocyclicrings, heterocyclic rings, or combinations thereof. The combinations canbe fused or covalently linked. Within certain preferred embodiments R3is a 2,5 disubstituted aryl. Preferably the substitutions are aryl oralkyl. Within a preferred embodiment R3 is 2,6-di-t-butyl-phenyl.

R4 and R5 are each independently hydrogen, alkyl groups having from 1 to6 carbon atoms. Within certain embodiments R4 and R5 can be joinedtogether to form a ring which is a four-, five-, six- or seven-memberring, saturated or unsaturated. For those embodiments in which the ringis unsaturated, the ring can be an heteroaromatic ring (e.g., pyrimidyl,imidazyl). Within certain preferred embodiments R4 and R5 are hydrogen.

Z and X each independently represent either NH, NR, O, or S, in which Ris a lower alkyl group of from one to six carbon atoms. In preferredembodiments, z represents 0 and X represents NH. The symbols n and meach represent independently, integers from zero to six.

As used herein, the term “alkyl” refers to a saturated hydrocarbonradical which may be straight-chain or branched-chain (for example,ethyl, isopropyl, or t-butyl), or cyclic (for example cyclobutyl,cyclopropyl or cyclopentyl). Preferred alkyl groups are those containing1 to 6 carbon atoms.

The term “alkenyl” refers to an unsaturated hydrocarbon radical whichmay be a straight-chain, branched-chain or cyclic. Examples of alkenylsinclude vinyl, allyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl,3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and 5-hexenyl,as well as dienes and trienes of straight, branched or cyclic chains andthe like. Preferred alkenyl groups are those containing 1 to 6 carbonatoms.

The term “aryl” refers to an aromatic substituent which may be a singlering or multiple rings which are fused together, linked covalently orlinked to a common group such as an ethylene or methylene moiety. Thearomatic rings may each contain heteroatoms, preferred heteroatoms areN, S or O. Examples of aryl groups include phenyl, benzyl, naphthyl,biphenyl, diphenylmethyl, 2,2-diphenyl-1-ethyl, thienyl, pyridyl andquinoxalyl. Additionally, the aryl groups may be attached to other partsof the molecule at any position on the aryl radical which wouldotherwise be occupied by a hydrogen atom (such as, for example,2-pyridyl, 3-pyridyl and 4-pyridyl).

Heterocyclic rings contain at least one heteroatom selected from N, Oand S. Examples of carbocyclic and heterocyclic rings includecyclohexyl, cyclohexenyl, piperazinyl, pyrazinyl, morpholinyl,imidazolyl, triazolyl and thiazolyl.

The terms “substituted alkyl”, “substituted alkenyl”, “substitutedalkylaryl”, “substituted aryl”, “substituted carbocyclic ring”,“substituted heterocyclic ring” “substituted phenyl”, “substitutedbenzyl”, “substituted naphthylmethyl” and “substituted indolymethyl”refer to the above alkyl, alkenyl, alkylaryl, carbocyclic ring,heterocyclic ring, aryl, phenyl, benzyl, naphthylmethyl and indolymethylgroups substituted by one or more, preferably one, halogen, haloalkyl,hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino,dialkylamino, acyloxy, acyl, alkyl and aryl. Examples include4-ethoxybenzyl, 1-ethyl-indolylmethyl, 4-alloxybenzyl,1-allyl-indolylmethyl, 4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl or2-naphthylmethyl.

All numerical ranges in this specification and claims are intended to beinclusive of their upper and lower limits.

In one group of preferred embodiments, the calcitonin mimetics arerepresented by the formula:

In this formula, the symbols R1 and R2 have the meaning provided above.The symbols S1, S3 and S4 each independently represent a substituent onthe attached aromatic ring which is hydrogen, halogen, haloalkyl,hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino, monoalkylamino,dialkylamino, acyloxy, acyl, alkyl and aryl. The symbols S2 and S3 eachrepresent an aryl or alkyl. In certain preferred embodiments R1 issubstituted or unsubstituted phenyl, benzyl, naphthylmethyl orindolymethyl. R2 is an alkyl or alkenyl having from 1 to 6 carbon atoms,substituted or unsubstituted benzyl or naphthylmethyl. In certainparticularly preferred embodiments, S5 and S2 are t-butyl, R1 is4-ethoxybenzyl, 1-ethyl-indolylmethyl, benzyl, 4-alloxybenzyl,1-allyl-indolylmethyl, 4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl,2-naphthylmethyl or phenyl and R2 is ethyl, allyl, benzyl, or2-naphthylmethyl.

The calcitonin mimetics used in the present invention can be preparedusing commercially available materials. A general synthetic scheme forpreparing molecules of Formula I wherein R4 and R5 are hydrogen, usingmethodologies known in the art, is provided herein.

In a typical preparation, 100 mg of p-methylbenzhydrylamine (MBHA) resin(0.81 meq/g, 100-200 mesh) was contained within a sealed polypropylenemesh packet. Following neutralization with 5% diisopropylethylamine(DIEA) in dichloromethane (DCM), the resin was washed with DCM. Thefirst protected amino acid was coupled using hydroxybenzotriazole (HOBt)and diisopropylcarbodiimide (DICI) in DMF. Following removal of theamino protecting group, the mesh packet was shaken overnight in asolution of 0.1 M trityl chloride in DCM/DMF (9:1) in the presence ofDIEA. Completeness of the trityl coupling was verified using thebromophenol blue color test as described in Krchnak et al., (Coll.Czech. Chem. Commun. 53:2542, 1988, and repeated as necessary.

N-alkylation was then performed by treatment of the resin packet with 1M lithium t-butoxide in THF (20X) for 15 min, as described by D{graveover ( )}rner, et al., (Bioorg. Med. Chem. 4:709, 1996). Excess base wasthen removed by decantation, followed by addition of the individualalkylating agent in DMSO (20×, 0.1M). The solution was vigorously shakenfor 2 h at room temperature. This step is normally repeated three timesfor methyl iodide, and five times for the other alkylating agents. Smallaliquots of the resin can be cleaved to determine the completeness ofthis step. The trityl group was removed with 2% TFA in DCM (2×10 min).

The isocyanate of the incoming primary amine (or aniline) was performedby slowly adding a solution of the primary amine (0.3M in DCM, 24× overthe resin substitution) and DIEA (48×) dropwise to solution of 0.1Mtriphosgene (8X) in DCM. It is known in the art that the reaction doesnot proceed through the isocyanate for secondary amines. The packet waswashed, neutralized and the isocyanate solution added and shaken for 1hour at RT. Following decantation, the isocyanate solution was quenchedwith 10% NH₃ in DMF. The resin was washed with DCM, 0.05% NH3 in DMF,MeOH, DCM, and MeOH.

The product was cleaved from the resin with anhydrous HF by theprocedures of Houghten et al., (Int. J. Pep. Prot. Res. 27:673, 1986),in the presence of anisole. The product was extracted with 50% ACN/H₂Oand lyophilized, followed by relyophilization from 50% acetonitrile.

The compounds of the invention can be administered to warm bloodedanimals, including humans, to mimic the interaction of calcitonin withits receptor in vivo. Within one aspect, calcitonin mimetics of thepresent invention are contemplated to be advantageous for use intherapeutic defects for which calcitonin is useful. In particular, thecalcitonin mimetics are useful for the regulation of bone metabolism andreduction of serum calcium. The calcitonin mimetics of the invention canbe administered to warm blooded animals, including humans, to mimic theinteraction of calcitonin with its receptor in vivo. Thus, the presentinvention encompasses methods for therapeutic treatment of bone-relateddisorders. Such bone-related disorders include, but are not limited to,osteoporosis, Paget's Disease, hyperparathyroidism, osteomalacia,periodontal defects (bone loss), hypercalcemia of malignancy, idiopathichypercalcemia of infancy, and other related conditions. Calcitoninmimetics are also contemplated to be advantageous as analgesics, inparticular for relief of bone pain. Calcitonin mimetics are furthercontemplated to be advantageous in inhibiting bone resorption. Thecalcitonin mimetics of the present invention can also be used to inhibitgastric secretion in the treatment of acute pancreatitis andgastrointestinal disorders. The methods of the present invention may beused to treat these conditions in their acute or chronic stages.

Pharmaceutically or therapeutically effective amounts of calcitoninmimetics of the present invention can be formulated withpharmaceutically or therapeutically acceptable carriers for parenteral,oral, nasal, rectal, topical, transdermal administration or the like,according to conventional methods. Formulations may further include oneor more diluents, fillers, emulsifiers, preservatives, buffers,excipients, and the like, and maybe provided in such forms as liquids,powders, emulsions, suppositories, liposomes, transdermal patches andtablets, for example. Slow or extended-release delivery systems,including any of a number of biopolymers (biological-based systems),systems employing liposomes, and polymeric delivery systems, can also beutilized with the compositions described herein to provide a continuousor long-term source of the calcitonin mimetic. Such slow release systemsare applicable to formulations, for example, for oral, topical andparenteral use. The term “pharmaceutically or therapeutically acceptablecarrier” refers to a carrier medium which does not interfere with theeffectiveness of the biological activity of the active ingredients andwhich is not toxic to the host or patient. One skilled in the art mayformulate the compounds of the present invention in an appropriatemanner, and in accordance with accepted practices, such as thosedisclosed in Remington: The Science and Practice of Pharmacy, Gennaro,ed., Mack Publishing Co., Easton, Pa., 19th ed., 1995. Preferably suchcompounds would be administered orally or parenterally.

As used herein, a “pharmaceutically or therapeutically effective amount”of such a calcitonin mimetic is an amount sufficient to induce a desiredbiological result. The result can be alleviation of the signs, symptoms,or causes of a disease, or any other desired alteration of a biologicalsystem. For example, an effective amount of a calcitonin mimetic is thatwhich provides either subject relief of symptoms or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver. In particular, such an effective amount of a calcitoninmimetic results in reduction in serum calcium, inhibition of boneresorption, inhibition of gastric secretion or other beneficial effect.Effective amounts of the calcitonin mimetics can vary widely dependingon the disease or symptom to be treated. The amount of the mimetic to beadministered, and its concentration in the formulations, depends uponthe vehicle selected, route of administration, the potency of theparticular mimetic, the clinical condition of the patient, the sideeffects and the stability of the compound in the formulation. Thus, theclinician will employ the appropriate preparation containing theappropriate concentration in the formulation, as well as the amount offormulation administered, depending upon clinical experience with thepatient in question or with similar patients. Such amounts will depend,in part, on the particular condition to be treated, age, weight, andgeneral health of the patient, and other factors evident to thoseskilled in the art. Estimation of appropriate dosages effective for theindividual patient is well within the skill of the ordinary prescribingphysician or other appropriate health care practitioner. As a guide, theclinician can use conventionally available advice from a source such asthe Physician's Desk Reference, 48^(th) Edition, Medical Economics DataProduction Co., Montvale, N.J. 07645-1742 (1994). Typically a dose willbe in the range of 0.1-100 mg/kg of subject. Preferably 0.5-50 mg/kg.Doses for specific compounds may be determined from in vitro or ex vivostudies on experimental animals. Concentrations of compounds found to beeffective in vitro or ex vivo provide guidance for animal studies,wherein doses are calculated to provide similar concentrations at thesite of action.

Well established animal models are available to test in vivo efficacy ofcalcitonin mimetics. For example, the hypocalcemic rat model can be usedto determine the effect of synthetic calcitonin mimetics on serumcalcium, and the ovariectomized rat or mouse can be used as a modelsystem for osteoporosis. Bone changes seen in these models and in humansduring the early stages of estrogen deficiency are qualitativelysimilar. Calcitonin has been shown to be an effective agent for theprevention of bone loss in ovariectomized humans and also in rats(Mazzuoli, et al., Calcif. Tissue Int. 47:209-14, 1990; Wronski, et al.,Endocrinologv 129:2246-50, 1991).

Only those compounds which retain calcitonin-like activity, as assayedby a CRE-luciferase assay, for example, are within the scope of thisinvention. The calcitonin receptor is a member of the G-protein receptorfamily and transduces signal via activation of adenylate cyclase,leading to elevation of cellular cAMP levels (Lin, et al., Science254:1022-4, 1991). This assay system exploits the receptor's ability todetect other molecules, not calcitonin, that are able to stimulate thecalcitonin receptor and initiate signal transduction.

Receptor activation can be detected by: (1) measurement of adenylatecyclase activity (Salomon, et al., Anal. Biochem. 58:541-8, 1974;Alvarez and Daniels, Anal. Biochem. 187:98-103, 1990); (2) measurementof change in intracellular cAMP levels using conventionalradioimmunoassay methods (Steiner, et al., J. Biol. Chem. 247:1106-13,1972; Harper and Brooker, J. Cyc. Nucl. Res. 1:207-18, 1975); or (3) useof a CAMP scintillation proximity assay (SPA) method (Amersham Corp.,Arlington Heights, Ill.). While these methods provide sensitivity andaccuracy, they involve considerable sample processing prior to assay,are time consuming, may involve the use of radioisotopes, and would becumbersome for large scale screening assays.

An alternative assay system (described in WO96/31536) involves selectionof substances that are able to induce expression of a cyclic AMPresponse element (CRE)-luciferase reporter gene, as a consequence ofelevated cAMP levels or other signaling pathways, such as stimulation ofCa⁺⁺/Ip₃ pathway leading to CRE induction, in cells expressing acalcitonin receptor, but not in cells lacking calcitonin receptorexpression. Such cells could include, for example, Boris/KS10-3(expressing hamster calcitonin receptor and a CRE-luciferase reportergene in baby hamster kidney cells (BHK 570 cells)) or Hollex 1 or Hollex2 (expressing human calcitonin receptor and a CRE-luciferase reportergene in BHK cells, as described in W096/31536) or KZ10-20-48/pLJ6-4-25,which expresses the human glucagon receptor and a CRE-luciferasereporter gene in BHK cells. The human glucagon receptor is anothermember of the G-protein-coupled receptor that transduces signal throughadenylate cyclase-mediated elevation of cAMP. PTH can be used as acontrol as well.

This CRE-luciferase assay measures the end result of a multi-step signaltransduction pathway triggered when a calcitonin mimetic stimulates theG-coupled calcitonin receptor. The complexity of this pathway providesmultiple mechanisms for induction of luciferase transcription at pointsthat are downstream of the calcitonin receptor, and therefore may not becalcitonin receptor-specific (e.g., forskolin's direct activation ofadenylate cyclase). Any response triggered by non-specific inducers iseliminated by counter screening using the calcitonin receptor-negativecell lines described above.

The foregoing description and the following examples are offeredprimarily for illustration and not as limitations. It will be readilyapparent to those of ordinary skill in the art that the operatingconditions, materials, procedural steps and other parameters of thesystem described herein may be further modified or substituted invarious ways without departing from the spirit and scope of theinvention. The invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Preparation of Calcitonin Mimetics: Urea of Anilineand L-Leucine Methylamide

Preparation of Trityl-leucine Amide Resin

In the preparation of the urea of aniline and leucine methyl amide, 100mg of p-methylbenzhydrylamine (MBHA) resin (0.81 meq/g, 100-200 mesh)was contained within a sealed polypropylene mesh packet as used insimultaneous multiple synthesis as described in Houghten, (Proc. Natl.Acad. Sci. USA 82:5131, 1985). Following neutralization (1 min) with 5%diisopropylethylamine (DIEA) in dichloromethane (DCM) (3×5 ml), theresin was washed with DCM (3×5 ml). The resin packet was added to asolution of tert-butyloxycarbonyl-L-leucine (Boc-Leu) (111 mg, 0.48 meq)and 1-hydroxybenzotriazole (65 mg, 0.48 meq) in dimethylformamide (DMF)(2.4 ml) in a 10 ml polypropylene bottle. Following addition of 2.4 ml0.2 M diisopropylcarbodiimide (DICI) in DMF, the resin was shaken on areciprocation shaker for 1.5 h. The resin was then washed with DMF (3×5ml) and DCM (3×5 ml). The oc protecting group was then removed bytreatment with 55% trifluoroacetic acid in DCM for 30 min. The resin wasthen washed with DCM (2×5 ml), isopropanol (IPA, 3×5ml), DCM (3×5 ml),neutralized with 5%DIEA in DCM (3×5 ml), and washed with DCM (2×5 ml).The resin packet was then shaken overnight (16 h) in 5 ml of 0.1 Mtrityl chloride in DCM/DMF (9:1) in the presence of DIEA. Completenessof the trityl coupling was verified using the bromophenol blue colortest as described in Krchnak et al., (Coll. Czech. Chem. Commun.53:2542, 1988), and repeated as necessary.

N-Methylation of trityl-leucine Amide Resin

N-methylation was then performed by treatment of the resin packet with asolution of 3.2 ml 0.5 M lithium t-butoxide (LiOtBu) in THF for 15 min,as described by D{grave over ( )}rner, et al., (Bioorg. Med. Chem.4:709, 1996). Excess base was then removed by decantation, followed byaddition of a solution of 0.3 ml methyl iodide in 3.2 mldimethylsulfoxide (DMSO). The solution was shaken for 2 hours at roomtemperature. The solution was then removed, washed with THF (1×5 ml) andthe LiOtBu/methyl iodide treatment repeated. Following removal of thesolution, the resin was washed with DMF (3×5 ml), IPA (2×5 ml), DCM (3×5ml). The trityl group was removed by two treatments (10 min) with 2% TFAin DCM (5ml). The resin was then washed with DCM (2×5 ml), IPA (3×5 ml)and DCM (3×5 ml).

Preparation of the Urea of Aniline and Leucine Methylamide Resin

The resin packet was neutralized (1 min) with 5% DIEA in DCM (3×5 ml),and washed with DCM (2×5 ml). The isocyanate of aniline was thenperformed by slowly adding a solution of aniline (0.176 ml) in DCM (6.5ml) and DIEA (0.678 ml) dropwise with stirring to a solution oftriphosgene (193 mg) in DCM (6.5 ml). The resin packet was added to theisocyanate solution and shaken for 1 hour at room temperature. Followingdecantation, the resin was washed with DCM (1×5 ml), 0.05% NH3 in DMF(2×5 ml), IPA (1×5 ml), DCM (1×5 ml), and methanol (1×5 ml). The resinwas then dried under high vacuum overnight.

Cleavage of the Urea from the Resin

The product was cleaved from the resin using 5 ml of anhydrous HF for1.5 h at 0° C. by the procedures of Houghten et al., (Int. J. Pep. Prot.Res. 27:673, 1986). The product was extracted with 50% acetonitrile(ACN)/H₂O (2×5 ml) and lyophilized, followed by relyophilization from50% ACN/H₂0 (5 ml). 13.5 mg crude product (85% purity by RP-HPLC) havingthe expected molecular weight of 263 daltons was obtained.

Example 2

This example provides in vitro, ex vivo, and in vivo assays which can beused to evaluate compounds described herein for their use in therapeuticapplications.

Assays for Calcitonin Mimetic Activity

CRE-Luciferase Assay Method for Calcitonin Mimetics

Human calcitonin receptor-positive and receptor-negative BHK-570 (BabyHamster Kidney) cell lines were maintained by serial passage in growthmedium (DMEM supplemented with 10% heat-inactivated fetal calf serum(HI-FCS), 2 mM L-glutamine, 1 mM sodium pyruvate, 250 nM MTX, and 1mg/mL G418). On the day prior to assay, cells were trypsinized, adjustedto 2.5×10⁵ cells/ml in growth medium, plated in opaque white DynatechMicrolite microtiter tissue culture plates at 50 μL/well, and grownovernight to confluence (37° C., 5% CO₂ atmosphere).

Test substances were prepared in DMSO or 10% DMF at 100 times the finaldesired assay concentration. At the time of assay, test substances werediluted into assay medium to 100, 50, 25, and 12.5 μg/ml in DMEMsupplemented with 10% HI-FCS, 2 mM L-glutamine, 1 mM sodium pyruvate and20 mM Hepes, pH 7.25, then 50 μl/well was added to assay plates forfinal assay concentrations of 50, 25, 12.5 and 6.25 μg/ml in 1% DMSO (or0.1% DMF). Controls were included on each plate: untreated wells(basal), 25 mM forskolin, and 100 nM human calcitonin. DMSO or DMF wasincluded in control wells at a concentration equal to that in testsamples (not to exceed a final assay concentration of 2% DMSO or 0.5%DMF, with a preferred maximum of 1% DMSO or 0.1% DMF).

Plates were incubated for 3 to 8 hours (4 hours preferred) at 37° C. inan atmosphere of 5% CO₂. Following induction, luciferase activity wasmeasured using a Promega luciferase assay kit (E1500) according to theassay kit protocol (Promega Corp., Madison, Wis.). Briefly, assay mediumwas removed and cells were washed once with phosphate buffered saline(PBS). After the wash, 25 μl of lysis buffer was added to each well, andthe plates were incubated for 15 minutes at room temperature. Fiftymicroliters of Luciferase Assay Substrate (Promega, Corp.) was added toeach well and the plates were transferred to a Labsystems Lumiscanmicrotiter luminometer (Labsystems Inc., Morton Grove, IL). The amountof luminescence (relative light units, RLU) was determined following a0.1 second/well integration of signal. Basal (uninduced) luciferasesignal was subtracted from all measurements, and the luciferase signalinduced by test samples was expressed as a percentage of the signal inthe calcitonin and forskolin controls. Specificity of the luciferaseinduction for calcitonin receptor-positive cell lines was determined bycomparing the percent control values in the calcitonin receptor-positiveline (Hollex-1) to those observed in the calcitonin receptor-negativecell line (KZ10-20-48/Zem 228) and the PTH receptor-positive cell line(KZ10-20-48/PTH-20) described below. Samples inducing a signal over thebasal level were selected for further characterization, see Table 1 forexamples.

TABLE 1 Luciferase induction (% of maximum luciferase induction producedby CT, 100 nM)

Crude μg/ml TPI # R1 R2 50 25 12.5 6.25 3.125 628-007

31.73 55.34 31.97 10.61 0.76 628-008

50.39 50.93 20.07 2.91 −1.31 628-013

64.26 69.62 51.62 17.98 3.51 628-015

71.49 72.17 43.2 14.69 1.62 628-016

75.76 80.63 76.44 43.89 10.72 628-022

46.98 41.52 17.85 3.26 2.08 628-023

41.79 46.84 32.04 7.84 −0.46 628-024

30.25 22.64 11.8 1.39 −1.24 628-025

22.61 7.96 2.33 0.65 0.42 628-042

60.41 68.69 59.74 49.17 19.16 628-043

47.95 46.24 42.06 34.53 6.48 628-044

30.14 26.23 29.35 24.08 10.65 628-045

40.62 44.51 42.49 38.73 20.19 628-052

38.27 40.21 36.63 23.59 9.57 628-053

40.14 41.8 32.31 11.89 3.29 628-054

54.86 47.47 42.54 27.05 10.73 628-056

37.1 35.6 28.6 14.7 4.2 TPI # R1 R2 Purified μg/ml 628-055

48.69 43.3 35.48 24.81 628-032

33.94 31.34 27.28 25.42 628-033

33.62 31.63 27.68 14.71 628-034

38.68 33.86 25.02 6.89 628-035

52.27 46.43 43.25 34.84 628-036

10.38 8.0 6.43 4.12

Test substances that appear to specifically elevate luciferaseexpression in CT-R positive cells but not CT-R negative cells weresubjected to an additional specificity check, i.e. their inability toactivate other members of the G-protein coupled receptor family. Theparathyroid hormone (PTH) receptor is another member of the G-proteincoupled receptor family that transduces signal through adenylate cyclasemediated elevation of cAMP. The receptor negative CRE-luciferase/DHFRexpressing BHK570 clone (KZ10-20-48) was transfected with the plasmidphupthr.2, encoding the cloned human PTH receptor in plasmid pHZ-1 whichalso contains the G418 selectable marker. Stable transfectants wereselected in 250 nM MTX+1 mg/ml G418 and were screened for CRE-luciferaseinduction in response to 25 mM forskolin or 100 nM human PTH (Sigma) (asdescribed in WO96/31536). Clone KZ10-20-48/PTH-20 was selected for usein specificity confirmation. This clone exhibits a 25 fold induction ofluciferase in response to human PTH (EC50=0.02 nM ) or forskolin(EC50=2.0 uM).

Calvarial Assay

Calvaria from 4-day old neonatal CD-1 mice (pregnant mice received fromCharles River Laboratories, Wilmington, Mass.) were trimmed withfine-tipped scissors to leave the parietal regions, including thesagittal suture. These trimmed bones were placed singly per well into6-well cell culture cluster plates (Costar, Pleasanton, Calif.) with 1ml/well of Dulbecco's Minimum Essential Medium, 4.5 ug/ml glucose (DMEM,BioWhittaker, Walkersville, Md.) or Basal Eagle's Medium with Earle'ssalts (EMEM, Gibco/BRL, Grand Island, N.Y.) and 0.29 mg/ml L-glutamine,1 mM sodium pyruvate, 15% heat-inactivated horse serum, and antibiotics(penicillin-G 50 μg/ml, streptomycin 50 μg/ml, and neomycin 100 μg/ml).Calvaria were rocked gently (RedRocker™, model PR50-115V, Hoefer, SanFrancisco, Calif. or Labline Rocking Shaker, model 4635, LablineInstruments, Melrose Park, Ill.) at 37° C. in a 5% CO₂ humidifiedincubator for 24 hours preincubation.

Following preincubation, medium was removed and replaced with 1.5ml/well of growth medium containing 1 nM parathyroid hormone (PTH) 1-34(Sigma) to stimulate bone resorption. For evaluation of the ability ofcalcitonin mimetics to inhibit PTH induced bone resorption, mimeticcompounds in DMSO were added to the growth medium at concentrationsranging from 1-400 μg/ml (final assay concentration of DMSO less than orequal to 1%). In each experiment human calcitonin (0.02-2OnM, 0.2-2nMpreferred) was added to PTH treated bones as a positive control. Controlwells that did not receive PTH, human calcitonin or calcitonin mimeticwere included for determination of calcium release from untreated bones.All control wells contained a final assay concentration of DMSO equal tothat present in calcitonin mimetic treated wells.

Five bones were included in each sample group. Bones were incubated for72 hours following PTH addition to allow resorption of bone to occur.Observations were made of the general appearance, healthiness and numberof cells that migrate from the calvaria during the incubation as apossible indication of cell toxicity. Calvaria to be examinedhistologically were transferred to glass scintillation vials containing10 ml of 10% neutral buffered formalin.

The medium was removed from the wells, and total calcium measurementswere made using a Nova 7/7+7 Electrolyte Analyzer or Nova CRT 10analyzer (Nova Biomedical, Waltham, Mass.) according to themanufacturer's specifications. Induction of bone resorption by PTH isseen as an increase in the concentration of calcium in the growth mediumdue to degradation of the bone matrix. Human calcitonin and biologicallyactive calcitonin mimetics inhibit this bone resorptive process asdemonstrated by a lowering of the calcium in growth medium as comparedto bones treated with PTH alone.

Calvaria Histology

To confirm the findings in the calvarial bone resorption assay employingcalcium release from culture mouse calvariae, selected bones were fixedin 10% neutral buffered formalin and demineralized in 5% formic acidwith 5% formalin. The bones were dehydrated through an ascending seriesof ethanol concentrations, infiltrated in glycol methacrylate, andembedded using a JB-4 embedding kit (PolySciences, Warrington, Pa.)(Liu, et al., J. Bone Mineral Res. 5:973-82, 1990). When necessary, analternative embedding method (paraffin embedding) was used to speed upthe embedding process. Cross sections of calvariae cut at 5 μm wereobtained and stained for tartrate-resistant acid phosphatase (TRAP)activity and counterstained with methyl green and thionin for cellmorphology (Liu, et al., ibid.). Osteoclasts were identified by TRAPstain, multinucleation, large cell size, and irregular cell shape. Thenumber of osteoclasts were counted from endocranial and ectocranial bonesurfaces and expressed as number/mm perimeter. The size of all theosteoclasts counted was also measured using a Bone Morphometry program(Liu, et al., ibid.; Bain, et al., J. Bone Miner. Res. 8:435-42, 1993).This histomorphometric method demonstrated increases in the number andsize of osteoclasts due to human parathyroid hormone (PTH 1-34)treatment. This PTH-induced increase was suppressed by treatment withhuman calcitonin.

Calcitonin mimetic compounds were evaluated in a similar fashion fortheir ability to suppress PTH-induced increases in osteoclast number andsize (Table 2). Cell toxicity (or death) was also evaluated by theappearance of pyknotic nuclei in a small number of bone cells. With anincreased level of toxicity, a further increase in the number of thesepyknotic nuclei, detachment of cells from bone surfaces, and losses ofcytoplasmic stain and cell boundaries were observed. The osteocyticspace also appeared empty.

TABLE 2 Effect of Calcitonin Mimetics on PTH-Induced Bone Resorption inMouse Calvariae Release of PTH Induced Ca++ Calcitonin Mimetic IC50(μg/ml) Histology 628-033 30 Decreases in bone destruction by PTH, Ocsize and # 628-035 23 Decreases in bone destruction by PTH, Oc size and# 628-055 14 Decreases in bone destruction by PTH, Oc size and # Oc =Osteoclast For comparative purposes, the IC50 for human calcitonin isabout 0.2 to 0.5 nM.

There was no apparent toxicity or tissue necrosis detected based onhistological observation of calvaria treated with up to 50 ug/ml ofcompound.

Induction of Hypocalcemia in Rats

This assay is based on the in vivo acute effect of calcitonin onosteoclasts, which causes rapid retraction of osteoclasts from bonesurface (typically within 30 minutes) and which results in decreasedbone resorption. See Mills, et al., in Endocrinology 1971—Proceedings ofthe Third International Symposium, Taylor (ed), Heinemann Medical,London, pp. 79-88 (1972) and Singer, et al., Clin. Endocrinol.5(Supp):333s-40s, 1976. The assay method was modified from the methoddescribed by Sturtridge and Kumar, Lancet 545:725-6, 1968. For the assayof hypocalcemic activity, weanling male Holtzman Sprague-Dawley rats (22days old) are infused with vehicle (PBS with 1 mM HCl and 0.1% BSA),calcitonin or calcitonin mimetics through the tail vein. One hour later,blood samples are collected by orbital sinus puncture to determine serumlevels of calcium. A decrease in serum calcium indicates a hypocalcemicresponse. The hypocalcemic response is dose-dependent as determinedusing salmon calcitonin (0.5, 2.5, 5, 50 and 100 ng/rat) in this model.

Inhibition of PTH-Induced Hypercalcemia in TPTX Rats

Continuous PTH infusion is associated with extensive destruction andsevere hypercalcemia in thyroparathyroidectomized (TPTX) rats. SeeThompson, et al., Proc. Natl. Acad. Sci. USA 85:5673-7, 1988. An animalmodel has been successfully established. See Liu, et al., J. BoneMineral Res. 11 (Suppl. 1):S206, 1996. For in vivo assay, maleSprague-Dawley rats (weighing about 150 g) are thyroparathyroidectomizedand the success of surgery is determined by measuring the levels ofserum calcium. Animals which are successfully operated on (serum calciumlevels less than 8 mg/dl) are maintained on a low calcium diet (0.02% Caand 0.6% P, ICN special diet) and infused s.c. with vehicle (PBS with 1mM HCl and 0.1% BSA), PTH (75 ug human PTH 1-34/kg body weight/day),PTH+calcitonin (salmon calcitonin 50 U/kg body weight/day), orPTH+calcitonin mimetic via Alzet osmotic minipumps (Model 1003D, AlzaCorp., Palo Alto, Calif.). Two days after infusion, animals aresacrificed and blood samples are collected to determine if thehypercalcemic response induced by PTH is inhibited by co-administrationof calcitonin or calcitonin mimetic. Additionally, tibial and kidneysamples are collected to determine osteoclastic bone resorption andnephrocalcinosis, respectively, and to confirm the findings in serumchemistry. Severe hypercalcemia induced by PTH has been shown to beaccompanied by increases in the number and size of osteoclasts,extensive bone destruction, and calcification in kidneys(nephrocalcinosis) following only two days of treatment (see, Liu, etal., ibid.). The serum, bone and kidney changes were attenuated byco-administration of CT.

Bone loss induced by combined ovariectomy and immobilization in rats

Estrogen deficiency and immobilization both induce bone loss in humansand in experimental animals. The combined effects cause severeosteopenia. See, Strachan, et al., J. Bone Mineral Res. 11 (Suppl.1):S456, 1996. A few studies have also shown that calcitonin iseffective at reducing bone loss associated with combined ovariectomy andimmobilization. See, Hayashi, et al., Bone 10:25-8, 1989 and McSheehy,et al., Bone 16:435-44, 1995. Slightly modified procedures were recentlyused to reproduce those results and demonstrate that calcitonin is veryeffective at reducing bone loss associated with the combined surgery,when evaluated by PQCT or histomorphometry in rats (see, Strachan, etal., ibid.).

For induction of bone loss, 2-month old Sprague-Dawley rats (weighingabout 200 g) are ovariectomized and immobilized by neurotomy of thesciatic nerve in the left hind limb. The immobilized animals are treatedwith vehicle (PBS with 1 mM HCl and 0.1 BSA), calcitonin (15 U/kg bodyweight/day),or calcitonin mimetics for 6 weeks. Calcein injections (15mg/kg body weight/day) are given i.p. at 9 and 2 days prior tosacrifice. Bone histomorphometry is performed as previously described(see, Liu, et al., J. Bone Mineral Res. 5:973-82, 1990) to determine theeffects of calcitonin and calcitonin mimetics.

Calvarial Assay to Determine Calcitonin Escape

Calvaria from 4-day old neonatal CD-1 mice (pregnant mice received fromCharles River Laboratories) are trimmed with fine-tipped scissors toleave the parietal regions, including the sagittal suture. These trimmedbones are placed singly per well into 6-well culture cluster plates(Costar) with 1 ml/well of growth medium, (Eagle's with Earle's salts(GIBCO BRL) containing 4.5 g/l glucose, 0.29 mg/ml L-glutamine, 1 mMsodium pyruvate, 15% heat-inactivated horse serum, antibiotics(penicillin-G 50 μg/ml, streptomycin 50 μg/ml, and neomycin 100 μg/ml)and 5 nM parathyroid hormone (PTH) 1-34 (Sigma)), and rocked gently(RedRocker™) at 37° C. in a 5% CO₂ humidified incubator for 17.5 hourspreincubation. The concentration of PTH is chosen to insure maximumresorption.

Following preincubation, medium is removed and replaced with 1 ml/wellof growth medium, as above, containing 20 or 30 μg/ml of the calcitoninmimetic in DMSO (final assay concentration of DMSO less than or equal to1%). Positive controls which can be used include, growth mediumcontaining 0.5, 1.0, 5.0 or 10 nM human calcitonin (hCT) and/or 0.01,0.02, 0.05 or 0.2 nM salmon calcitonin (sCT). Control wells that do notreceive PTH, human or salmon calcitonin or the calcitonin mimetic areincluded for determination of calcium release from untreated bones. Allcontrol wells contain a final assay concentration of DMSO equal to thatpresent in the calcitonin mimetic treated wells.

Five bones are included in each sample group. Bones are incubated for 4,8, 11, 24, 50.5, 72.5 and 98 hours. At each time point the media isremoved and fresh dilutions of compound in media are added to thecalvaria. After the media is removed, total calcium measurements aremade using a Nova 7/7+7 Electrolyte Analyzed (Nova Biomedical) accordingto the manufacturer's specifications. Induction of bone resorption byPTH is seen as an increase in the concentration of calcium in the growthmedium due to degradation of the bone matrix.

Human and salmon calcitonin and biologically active calcitonin mimeticsinhibit the bone resorptive process as demonstrated by a lowering of thecalcium in growth medium as compared to bones treated with PTH alone.The inhibitory effect of hCT and sCT is lost after a period of time,generally 24 hours, and the rate of resorption follows the same slope asthat of PTH alone. Those mimetic compounds which do not have this escapewill be able to continue inhibition of resorption for a longer period oftime. Calvaria can also be observed, as described above, at each timepoint for signs of inhibition and signs of toxicity.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A compound having the formula:

wherein p1 R1 and R2 are each independently selected from the groupconsisting of hydrogen, alkyls having from 1 to 6 carbon atoms, alkenylshaving from 1 to 6 carbon atoms, aryl, substituted aryl, alkylaryl,substituted alkylaryl, carbocyclic ring, substituted carbocyclic ring,heterocyclic ring, substituted heterocyclic ring, and combinationsthereof, the combinations are fused or covalently linked and thesubstituents are selected from the group consisting of halogen,haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino,monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl; S1, S3 andS4 are hydrogen; and S2 and S5 are each independently alkyl or aryl. 2.A compound according to claim 1 wherein, R1 is selected from the groupconsisting of phenyl, substituted phenyl, benzyl, substituted benzyl,naphthylmethyl, substituted naphthylmethyl, indolymethyl, andsubstituted indolymethyl; R2 is selected from the group consisting ofalkyls having from 1 to 6 carbon atoms, alkenyls having from 1 to 6carbon atoms, benzyl, substituted benzyl, naphthylmethyl, andsubstituted naphthylmethyl; wherein the substituents are selected fromthe group consisting of halogen, haloalkyl, hydroxy, aryloxy, benzyloxy,alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl,alkyl and aryl; and S2 and S5 are t-butyl.
 3. A compound according toclaim 2 wherein, R1 is 4-ethoxybenzyl, 1-ethyl-indolylmethyl, benzyl,4-alloxybenzyl, 1-allyl-indolylmethyl, 4-chlorobenzyl, 4-flurobenzyl,4-iodobenzyl, 2-naphthylmethyl or phenyl; R2 is ethyl, allyl, benzyl or2-naphthylmethyl; and S2 and S5 are t-butyl.
 4. A pharmaceuticalcomposition comprising an effective amount of a compound having theformula:

wherein, R1 and R2 are each independently selected from the groupconsisting of hydrogen, alkyls having from 1 to 6 carbon atoms, alkenylshaving from 1 to 6 carbon atoms, aryl, substituted aryl, alkylaryl,substituted alkylaryl, carbocyclic ring, substituted carbocyclic ring,heterocyclic ring, substituted heterocyclic ring, and combinationsthereof, the combinations are fused or covalently linked and thesubstituents are selected from the group consisting of halogen,haloalkyl, hydroxy, aryloxy, benzyloxy, alkoxy, haloalkoxy, amino,monoalkylamino, dialkylamino, acyloxy, acyl, alkyl and aryl; S1, S3 andS4 are hydrogen; and S2 and S5 are each independently alkyl or aryl. 5.A pharmaceutical composition according to claim 4, wherein R1 isselected from the group consisting of phenyl, substituted phenyl,benzyl, substituted benzyl, naphthylmethyl, substituted naphthylmethyl,indolymethyl, and substituted indolymethyl; R2 is selected from thegroup consisting of alkyls of from 1 to 6 carbon atoms, alkenyls of from1 to 6 carbon atoms, benzyl, substituted benzyl, naphthylmethyl, andsubstituted naphthylmethyl; wherein the substituents are selected fromthe group consisting of halogen, haloalkyl, hydroxy, aryloxy, benzyloxy,alkoxy, haloalkoxy, amino, monoalkylamino, dialkylamino, acyloxy, acyl,alkyl and aryl; and S2 and S5 are t-butyl.
 6. A pharmaceuticalcomposition according to claim 4, wherein R1 is 4-ethoxybenzyl,1-ethyl-indolylmethyl, benzyl, 4-alloxybenzyl, 1-allyl-indolylmethyl,4-chlorobenzyl, 4-flurobenzyl, 4-iodobenzyl, 2-naphthylmethyl or phenyl;R2 is ethyl, allyl, benzyl or 2-naphthylmethyl; and S2 and S5 aret-butyl.